Method of identifying and treating invasive carcinomas

ABSTRACT

Prostasin protein has been found to be a useful marker for determination of the invasiveness of and as a means to treat human carcinomas. Using RT-PCR and western blot analyses, prostasin protein and mRNA expression were found in normal human prostate epithelial cells and the human prostate cancer cell line LNCaP, but not in the highly invasive human prostate cancer cell lines DU-145 and PC-3. Imunohistochemistry studies of human prostate cancer specimens revealed a down-regulation of prostasin in high-grade tumors. Using RT-PCR and western blot analyses, prostasin protein and mRNA expression were found in a non-invasive human breast cancer cell line, MCF-7, while invasive human breast cancer cell lines MDA-MB-231 and MDA-MB-435s were found not to express either the prostasin protein or the mRNA. A non-invasive human breast cancer cell line, MDA-MB-453, was shown to express prostasin mRNA but not prostasin protein. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68% and 42%, respectively. Transfection of MDA-MB-231 and MDA-MB-435s cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 50% for either cell line. The prostasin gene promoter region was found to be hypermethylated at specific sites in invasive cancer cells.

This invention relates to prostasin and its use in the diagnosis andtreatment of prostate and breast cancers, and is a Continuation-In-Part(CIP) of application Ser. No. 09/755,811 filed Jan. 5, 2001 now U.S.Pat. No. 6,569,684 and claims the priority to U.S. Provisionalapplication Ser. No. 60/174,801 filed Jan. 6, 2000 and was supported inpart by Department of Defense Prostate Cancer Research GrantDAMD17-98-1-8590, and in part by grants to principal investigator K. X.CHAI from the Florida Hospital Gala Endowed Program for OncologicResearch.

BACKGROUND AND PRIOR ART

For men in the U.S. prostate cancer is the most commonly diagnosedcancer, and the second leading cause of cancer-related death (Greenlee RT, Murray F. Bolden S, Wingo P A. Cancer statistics, 1999. Ca: a CancerJournal for Clinicians 2000;50:7-33). Prostate cancers originate aslocalized lesions; some of these localized lesions will progress tobecome invasive, migratory and metastatic. Our current understanding ofthe mechanisms of the prostate cancer invasion process, however, ispoor. Our ability to predict the acquisition of invasive potentials by aprostate cancer is limited.

The mechanisms leading to the development of a prostate cancer arecomplex. Currently, it is believed to be the result of multipletransformation steps from normal prostate glandular cells (Carter H B.Piantadosi S. Isaacs J T. Clinical evidence for and implications of themultistep development of prostate cancer. Journal of Urology.143(4):742-6, 1990). The initial steps result in what are described asprostatic interepithelial neoplastic (PIN) lesions (Isaacs J T.Molecular markers for prostate cancer metastasis. Developing diagnosticmethods for predicting the aggressiveness of prostate cancer. [Review][92 refs] American Journal of Pathology. 150(5):1511-21, 1997). ThesePIN lesions may then typically have three different fates based on anassessment of their impact to the patient. The PIN lesions can remain assuch, not producing histologically detectable prostate cancer, orfurther transform into histologically detectable prostate cancer. Mostof the histologically detectable prostate cancers will be asymptomaticin the patient and remain non-manifest clinically as many are discoveredpost-mortem (Carter H Coffey D. Prostate Cancer: the magnitude of theproblem in the United States. In a Multidisciplinary Analysis ofControversies in the Management of Prostate Cancer. (Eds. Coffey D.Resnick M. Door R. et al.), pp1-9, Plenum Press, 1988; Carter H BPiantadosi S. Issacs J T. Clinical evidence for implications of themultistep development of prostate cancer. Journal of Urology.143(4):742-6, 1990; Scardino P T. Weaver R. Hudson M A. Early detectionof prostate cancer. [Review] [102 refs] Human Pathology. 23(3):211-22,1999). Prostate cancers are diagnosed clinically by an estimate of sizeand location using the TNM staging system (Denis L J. Staging andprognosis of prostate cancer. European Urology. 24 Suppl 2:13-8, 1993),and by pathological staging based on an examination of the histology ofthe removed prostate via either biopsy or prostatectomy using a systemby D. F. Gleason, (Gleason D F. Classification of prostatic carcinomas.Cancer Chemotherapy Reports—Part 1. 50(3):125-8, 1966). About 50% ofprostate cancer cases receiving treatment are diagnosed clinically asadvanced, or, non-organ-confined (Scardino P T. Weaver R. Hudson M A.Early detection of prostate cancer. [Review] [102 refs] Human Pathology.23(3):211-22, 1992), for which no effective treatment exists (Yagoda A.Petrylak D. Cytotoxic chemotherapy for advanced hormone-resistantprostate cancer. [Review] [63 refs] Cancer. 71(3 Suppl): 1098-109, 1993and Petrylak, 1993). Of the remaining 50% cases, ⅓ (˜50,000) arediagnosed as organ-confined but micrometastasis may be present. Thefinal group of patients (˜100,000) have truly organ-confined prostatecancer and can be cured by radical prostatectomy (Sgrignoli A R Walsh PC. Steinberg G D. Steiner M S. Epstein J I. Prognostic factors in menwith stage D 1 prostate cancer: identification of patients less likelyto have prolonged survival after radical prostatectomy [see comments].Journal of Urology. 152(4):1077-81, 1994; Zincke H. Oesterling J E.Blute M L. Bergstralh E J. Myers R P. Barrett D M. long term (15 years)results after radical prostatectomy for clinically localized (stageT2cor lower) prostate cancer [see comments]. Journal of Urology.152(5 Pt2): 1850-7, 1994) or left untreated (watchful waiting) without the riskof life-threatening or life-altering. For the patients withnon-organ-confined prostate cancers (discovered via either biopsy orsurgery), undergoing systemic treatment early is essential to themanagement of their cancer (Yagoda A. Petrylak D. Cytotixic chemotherapyfor 1098-109, 1993). Consequently, it will be ideal, both medically andeconomically, if one could precisely predict upon early pathologicalexamination of the tumor, which group of patients will have trulyorgan-confined disease versus which group will have invasive prostatecancer.

Clinical staging of prostate cancer generally depends on the results ofthree tests that are performed in the following order: a PSA(prostate-specific antigen) blood test as a screening method; DRE(digital rectal examination) for an initial indication of palpabledisease; and, a biopsy to obtain samples for histological examination.Prostate cancers, removed either via biopsy or surgery, are gradedhistologically by the system of Gleason. (Gleason D F. Classification ofprostatic carcinomas. Cancer Chemotherapy Reports—Part1. 50(3):125-8,1966), which is an evaluation of how aggressive and howpoorly-differentiated the prostate cancers are. The aggressiveness ofprostate tumors: of low Gleason scores (<5) is limited; of high Gleasonscores (8-10) are highly aggressive; but, for the intermediateGleason-score (5-7) prostate cancers (76% of prostate tumors), theaccuracy of predicting their aggressiveness is poor (Gleason D F.Mellinger G T. Prediction of prognosis for prostatic adenocarcinoma bycombined histological grading and clinical staging. Journal of Urology.111(1):58-64, 1974). Thus, the ability to accurately determine theaggressiveness of these intermediate Gleason-score prostate tumors hasremained as a practical challenge to, and a primary goal for, prostatecancer research (Isaacs J T. Molecular markers for prostate cancermetastasis. Developing diagnostic methods for predicting theaggressiveness of prostate cancer. [Review] [92 refs] American Journalof Pathology. 150(5):1511-21, 1997). Especially with regard to thenumber of patients (150,000) facing a decision of whether to undergosystemic treatment, the most urgent demand in prostate cancer care isthe development of methods to enhance our ability to accurately predictthe aggressiveness of the tumors with Gleason scores of 5-7.

It is now commonly believed that cancers occur via multipletransformation steps by accumulating mutations in three classes ofgenes: proto-oncogenes (Park M. Oncogenes. In The Genetic Basis of HumanCancer (Eds. Vogelstein B and Kinzler K W), pp205-28, McGraw-Hill HealthProfessions Divisions, 1998); tumor-suppressor genes (Knuutila S. AaltoY. Bjorkqvist A M. EL-Rifai W. Hemmer S. Huhta T. Kettunen E.Kiuru-Kuhlefelt S. Larramendy ML. Lushnikova T. Monni O. Pere H. TapperJ. Tarkkanen M. Varis A. Wasenius V M. Wolf M. Zhu Y. DNA copy numberlosses in human neoplasms. [Review] [197 refs] American Journal ofPathology. 155(3):683-94, 1999); and, DNA repair genes (Knuutila S.Aslto Y. Bjorkqvit A M. EL-Rifai W. Hemmer S. Huhta T. Kettunen E.Kiuru-Kuhlefelt S. Larramedy M L. Lushnikova T. Monni O. Pere H. TapperJ. Tarkkanen M. Varis A. Wasenius V M. Wolf M. Zhu Y. DNA copy numberlosses in human neoplasms. [Review] [197 refs] American Journal ofPathology. 155(3):683-94, 1999). The histological prostate cancers forwhich the prediction of clinical aggressiveness is difficult (those withthe intermediate Gleason scores 5-7) probably have not gone through thenecessary “multi-step” transformation to acquire the potentials tobehave aggressively (as would the high-grade cancers). This notion wassupported by studies comparing the course of prostate cancer developmentamong men in Japan and in the U.S., and Japanese men who migrated to theU.S. (Carter H B. Piantadosi S. Isaacs J T. Clinical evidence for andimplications of the multistep development of prostate cancer. Journal ofUrology, 1990; Haenszel W. Kurihara M. Studies of Japanese Migrants. 1.Mortality form cancer and other diseases among Japanese in the UnitedStates. Journal of the National Cancer Institute.40(1):43-68, 1968;Akazaki K. Stemmerman G N. Comparartive study of latent carcinoma of theprostate among Japanese in Japan and Hawaii. Journal of the NationalCancer Institute. 50(5):1137-44, 1973; Dunn J E. Cancer epidemiology inpopulations of the United States—with emphasis on Hawaii andCalifornia—and Japan. Cancer Research. 35(11 Pt.2):3240-5, 1975). Thefindings were that first- and second-generation Japanese men whomigrated to the U.S. have a higher prostate cancer incident rate thannative Japanese men. The emigrant Japanese men's prostate cancerincident rate is similar to that of men in the U.S. Investigating thechanges of expression in these three classes of cancer-relevant genesduring the course of prostate cancer development will lead to a betterunderstanding of the processes by which prostate cancers acquire theiraggressive potentials. The discovery of molecules whose changes can becorrelated to the staging of prostate cancer will provide new tools toimprove our ability to better predict the aggressive behaviors ofprostate cancer. This approach is now commonly referred to as “molecularstaging”.

Down-regulated genes such as tumor suppressors, invasion suppressors ormetastasis suppressors may be used as prostate cancer markers. Examplesof these genes that can potentially serve as prostate cancer markers for“molecular staging” are: KAII (Dong J T. Suzuki H. Pin S S. Bova G S.,Schalken JA, Issacs W B, Barrett JC. Issace I T. Down-regulation of theKAI I metastasis suppressor gene during the progression of humanprostatic cancer infrequently involves gene mutation or allelic loss.Cancer Research. 56(19): 4387-90, 1996); (Ueda T, Ichikawa T., Tamaru j,Mikata A, Akakura K, Akimoto S, Imai T. Yoshie O. Shiraishi T. Yatani R.Ito H. Shimazaki J., Expression fof the KA11 protein in benign prostatichyperplasia and prostate cancer. American Journal of Pathology 149(5):1435-40, 1996); E-cadherin (Umbas R. Isaacs WB BringuierP P. Schaafsma HE. Karthaus H F. Oosterhof G O. Debruyne F M. Schalken J A. DecreasedE-Cadherin expression is associated with poor prognosis is patients withprostate cancer. Cancer Research. 52(18):5104-9, 1992, Umbas R. Isaacs WB. Bringuier P P. Schaafsma H E. Karthaus H F. Oosterhof G O. Debruyne FM. Schalken J A. Decreased E-cadherin expression is associated with poorprognosis in patients with prostate cancer. Cancer Research.54(14):3929-33, 1994); β_(1C) integrin (Formaro M. Tallini G. BofetiadoC J. Bosari S. Languino L R. Down-regulation of β1C integrin, aninhibitor of cell proliferation, in prostate carcinoma. American Journalof Pathology. 149(3):765-73,1996 Formaro M. Manzotti M. Tallini G. StearA E. Bosari S. Ruoslahti E. Languino L R. β1C integrin in epithelialcells correlates with a nonproliferative phenotype: forced expression ofβ1C inhibits prostate epithelial cell proliferation. American Journal ofPathology. 153(4):1079-87, 1998; p27(kip1) (Tsihlias J. Kapusta LR.DeBoer G. Morava-Protzner I. Zbieranowski I. Bhattacharya N. CatzavelosG C. Klotz L H. Slingerland J M. Loss of cyclin-dependent kinaseinhibitor p27Kip1 is a novel prognostic factor in localized humanprostate adenocarcinoma. Cancer Research. 58(3):542-8, 1998); and CD44(Lou W. Krill D. Dhir R. Becich M J. Dong J T. Frierson H F Jr. Isaacs WB. Isaacs J T. Gao A C. Methylation of the CD44 metastasis suppressorgene in human prostate cancer. Cancer Research. 59(10):2329-31, 1999).By using the method of immunohistochemistry, these genes were found tobe down-regulated in prostate cancer. KAI's down regulation is apotential predictor of metastasis (Dong, et al., 1996: Ueda et al.,1996). While E-cadherin'cadherin's down regulation is stronglycorrelated to higher Gleason grades. Functional studies of these geneshave given clues to their role in prostate cancer or normal prostatebiology. (Debruyne F M. Isaacs W B. Expression of the cellular adhesionmolecule E-cadherin is reduced or absent in high-grade prostate cancer.Cancer Research. 52(18):5104-9, 1992; Umbas R. Isaacs W B. Bringuier PP. Schaafsma H E. Karthaus H F. Oosterhof G O. Debruyne F M. Schalken JA. Decreased E-cadherin expression is associated with poor prognosis inpatients with prostate cancer. Cancer Research. 54(14):3929-33, 1994;metastasis suppressor gene during the progression of human prostaticcancer infrequently involves gene mutation or allelic loss. CancerResearch. 56(19):4387-90; 1996 Ueda T. Ichikawa T. Tamaru J. Mikata A.Akakura K. Akimoto S. Imai T. Yoshie O. Shiraishi T. Yatani R. Ito H.Shimazaki J. Expression of the KAI1 protein in benign prostatichyperplasia and prostate cancer. American Journal of Pathology. 149(5):1435-40, 1996) while E-cadherin's down-regulation is strongly correlatedto higher Gleason grades (Umbas R. Schalken J A. Aalders T W. Carter BS. Karthaus H F. Schaafsma H E. Debruyne F M. Isaacs W B. Expression ofthe cellular adhesion molecule E-cadherin is reduced or absent inhigh-grade prostate cancer. Cancer Research. 52(18):5104-9,1992; UmbasR. Isaacs W B. Bringuier P P. Schaafsma H E. Karthaus H F. Oosterhof GO. Debruyne F M. Schalken J A. Decreased E-cadherin expression isassociated with poor prognosis in patients with prostate cancer. CancerResearch. 54(14):3929-33, 1994) Functional studies of these genes havegiven clues to their role in prostate cancer or normal prostate biology.Human KAI suppress metastasis of rat prostate cancer cells upon genetransfer (Dong J T. Lamb P W. Rinker-Schaeffer C W. Vukanovic J.Ichikawa T. Isaacs J T. Barrett J C. KAI1, a metastasis suppressor genefor prostate cancer on human chromosome 11p11.2 [see comments]. Science.268(5212):884-6,1995). β_(1C) and p27(kip1) are involved in signalingpathway that inhibits cell proliferation (Formaro M. Tallini G. Zheng DQ. Flanagan W M. Manzotti M. Languino L R. p27(kipl) acts as adownstream effector of and is coexpressed with the beta1C integrin inprostatic adenocarcinoma. Journal of Clinical Investigation.103(3):321-9, 1999). E-cadherin expression is progressively lost duringthe transformation of rat prostate cancer from non-invasive to invasive(Bussemakers M J. van Moorselaar R J. Giroldi L A. Ichikawa T. Isaacs JT. Takeichi M. Debruyne F M. Schalken J A. Decreased expression ofE-cadherin in the progression of rat prostatic cancer. Cancer Research.52(10):2916-22, 1992), and it has also been shown to be an invasionsuppressor (Lou W. Krill D. Dhir R. Becich M J. Dong J T. Frierson H FJr. Isaacs W B. Isaacs J T. Gao A C. Methylation of the CD44 metastasissuppressor gene in human prostate cancer. Cancer Research.59(10):2329-31, 1999). CD44 loss of expression is associated with highmetastatic ability and transfection of CD44 suppresses metastasiswithout affecting tumorigenecity of rat prostate cancer cells (Gao A C.Lou W. Dong J T. Isaacs J T. CD44 is a metastasis suppressor gene forprostatic cancer located on human chromosome 11p13. Cancer Research.57(5):846-9, 1997). CD44 down-regulation is a prognostic marker forprostate cancer (Noordzij M A. van Steenbrugge G J. Verkaik N S.Schroder F H. van der Kwast T H. The prognostic value of CD44 isoformsin prostate cancer patients treated by radical prostatectomy. ClinicalCancer Research. 3(5):805-15, 1997). Human prostate cancer histology isheterogeneous, or “multi-focal” in nature (Isaacs JT. Bova GS. ProstateCancer. In The Genetic Basis of Human Cancer (Eds. Vogelstein B andKinzler KW), pp653-60, McGraw-Hill Health Professions Division, 1998 andBova, 1998), with an average of five lesions in a patient (Bastacky S I.Wojno K J. Walsh PC. Carmichael M J. Epstein J I. Pathological featuresof hereditary prostate cancer. Journal of Urology. 153(3 Pt 2):987-92,1995). Between the tumor regions of a prostate and within a single tumorregion, the genetic causes to the cancer are heterogeneous andindependent as well (Sakr W A. Macoska J A. Benson P. Grignon D J.Wolman S R. Pontes J E. Crissman J D. Allelic loss in locallymetastatic, multisampled prostate cancer. Cancer Research.54(12):3273-7, 1994; Qian J. Bostwick D G. Takahashi S. Borell T J.Herath J F. Lieber M M. Jenkins R B. Chromosomal anomalies in prostaticintraepithelial neoplasia and carcinoma detected by fluorescence in situhybridization. Cancer Research. 55(22):5408-14, 1995; Mirchandani D.Zheng 1. Miller G J. Ghosh A K. Shibata D K. Cote R J. Roy-Burman P.Heterogeneity in intratumor distribution of p53 mutations in humanprostate cancer. American Journal of Pathology. 147(1):92-101, 1995). Inthe end, the best predictor of prostate cancer's potential to gaininvasiveness may be a consideration of a number of genes whoseexpression levels change along the course of cancer development, asdemonstrated in principle by Greene et al. (Greene G F. Kitadai Y.Pettaway C A. von Eschenbach A C. Buucana C D. Fidler I J. Correlationof metastasis-related gene expression with metastatic potential in humanprostate carcinoma cells implanted in nude mice using an in situmessenger RNA hybridization technique. American Journal of Pathology.150(5):1571-82, 1997). Hence, expanding the repertory of such genes,including both the onco-genes and the suppressor genes, will enhance theaccuracy and dependability of this approach. As for the treatmentoptions of prostate cancer, patients with truly organ-confined prostatecancer can be cured by radical prostatectomy. Patients withnon-organ-confined prostate cancers, however, have very low survivalrates and the current treatments are largely ineffective (Yagoda A.Petrylak D. Cytotoxic chemotherapy for advanced hormone-resistantprostate cancer. [Review] [63 refs] Cancer. 71(3 Suppl): 1098-109,1993). Breast cancer is the most diagnosed cancer in women and thesecond leading cancer related cause of death in women (Greenlee R T,Murray T, Bolden S, Wingo P A. Cancer statistics, 1999. Ca: a CancerJournal for Clinicians 2000;50:7-33). Breast ductal carcinoma in situ(hereinafter indicated as DCIS) incidence has increased dramaticallysince 1983 as a result of implementing screening programs (Ernster V L.Barclay J. Increases in ductal carcinoma in situ (DCIS) of the breast inrelation to mammography: a dilemma. [Review] [34 refs] Journal of theNational Cancer Institute. Monographs.

(22): 151-6, 1997). DCIS is described as a malignant growth ofepithelial cells within the ducts and lobules of the breast, and isbelieved to be the precursor of all invasive breast carcinoma. DCISitself is non-life-threatening; however, current treatment options forDCIS include masectomy, lumpectomy, radiotherapy or tamoxifen (Ernster VL. Barclay J. Increases in ductal carcinoma in situ (DCIS) of the breastin relation to mammography: a dilemma. [Review] [34 refs] Journal of theNational Cancer Institute. Monographs. (22): 151-6, 1997; Hwang E S.Esserman L J. Management of ductal carcinoma in situ. [Review] [91 refs]Surgical Clinics of North America. 79(5):1007-30, viii, 1999). Thesetreatment options for DCIS are at best controversial due primarily to alack of precision in diagnosis and prognosis of whether the detectedDCIS will progress to invasive breast cancer and whether recurrence islikely after treatment, usually with a high percentage being invasivebreast cancer (Zaugg K. Bodis S. Is there a role for molecularprognostic factors in the clinical management of ductal carcinoma insitu (DCIS) of the breast?. [Review] [49 refs] Radiotherapy & Oncology.55(2):95-9, 2000). At present, histological grading (nuclear grading andwhether come do-type necrosis is present) and the size of the DCIS areused to provide assessments of risk of DCIS to progress into invasivebreast cancer (Zaugg K. Bodis S. Is there a role for molecularprognostic factors in the clinical management of ductal carcinoma insitu (DCIS) of the breast?. [Review] [49 refs] Radiotherapy & Oncology.55(2):95-9, 2000; Shoker BS. Sloane JP. DCIS grading schemes andclinical implications. [Review] [40 refs] Histopathology. 35(5):393-400,19; van de Vijver M J. Ductal carcinoma in situ of the breast:histological classification and genetic alterations. [Review] 169 refs]Recent Results in Cancer Research. 152:123-34, 1998). These parametersare far from being adequate for making the most accurate choice oftreatment, resulting in a choice either overly excessive orconservative, in either case, the patient will suffer unnecessarily.Molecular markers can help improve our ability to better diagnose DCISand stratify treatment options, especially the molecular markers that,themselves, play a role in the progression of DCIS to invasive breastcancer (Zaugg K. Bodis S. Is there a role for molecular prognosticfactors in the clinical management of ductal carcinoma in situ (DCIS) ofthe breast?. [Review] [49 refs] Radiotherapy & Oncology. 55(2):95-9,2000; Silverstein M J. Masetti R. Hypothesis and practice: are thereseveral types of treatment for ductal carcinoma in situ of the breast?.[Review] [53 refs] Recent Results in Cancer Research. 152:105-22, 1998).The tumorigenesis process is a multi-step transformation in whichmolecular events escalate to the final stage of invasive phenotype(Silverstein MJ. Masetti R. Hypothesis and practice: are there severaltypes of treatment for ductal carcinoma in situ of the breast?. [Review][53 refs] Recent Results in Cancer Research. 152:105-22, 1998). Theconventional paradigm of protease involvement in the development andprogression of cancer has been the assignment of a usually negative roleto the proteases, such as promoting tumor invasion (Mignatti P. Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. [Review][306 refs] Physiological Reviews. 73(1):161-95, 1993). In turn, theconventional paradigm of protease inhibitors in relation to cancer isusually regard of a beneficial effect for the presence of thesemolecules (Kennedy AR. Chemopreventive agents: protease inhibitors.Pharmacol Therapeut 78:167-209, 1998). Recently, however, the picture ofa new paradigm is beginning to emerge for several serine proteases inbreast, prostate, and testicular cancers. A “normal epithelial cellspecific-1” (NES1) serine protease was found to be down-regulated inbreast and prostate cancers, and it functions as a tumor suppressor(Goyal J, Smith K M, Cowan J M, Wazer D E, Lee S W, Band V. The role forNES1 serine protease as a novel tumor suppressor. Cancer Res58:4782-4786, 1998). A prostate-specific serine protease, prostase(Nelson P S. Gan L. Ferguson C. Moss P. Gelinas R. Hood L. Wang K.Molecular cloning and characterization of prostase, anandrogen-regulated serine protease with prostate-restricted expression.Proceedings of the National Academy of Sciences of the United States ofAmerica. 96(6):3114-9, 1999), was shown to be expressed in normalprostate but not in prostate cancer cell lines DU-145 and PC-3. Theexpression of a testis-specific serine protease, testisin, was shown tobe lost in testicular cancer through either a loss of gene (Hooper J D.Nicol D L. Dickinson J L. Eyre H J. Scarman A L. Normyle J F. Stuttgen MA. Douglas M L. Loveland K A. Sutherland G R. Antalis T M. Testisin, anew human serine proteinase expressed by premeiotic testicular germcells and lost in testicular germ cell tumors. Cancer Research.59(13):3199-205, 1999) or methylation in the promoter (Boucaut K,Douglas M, Clements J, Antalis T. The serine proteinase testisin may actas a tumor and/or growth suppressor in the testis and may be regulatedby DNA methylation. Cancer Genetics and Tumor Suppressor Genes, ColdSpring Harbor Laboratory, 2000). Further, transfection of humantesticular cancer cells with a testisin cDNA reduced the tumor growth ofxenografts of these cells in nude mice, suggesting a tumor suppressorfunction for testisin (Boucaut K, Douglas M, Clements J, Antalis T. Theserine proteinase testisin may act as a tumor and/or growth suppressorin the testis and may be regulated by DNA methylation. Cancer Geneticsand Tumor Suppressor Genes, Cold Spring Harbor Laboratory, 2000). Thetestisin serine protease is potentially membrane-bound as suggested byits structure and confirmed by immunohistochemistry gene (Hooper J D.Nicol D L. Dickinson J L. Eyre H J. Scarman A L. Normyle J F. Stungen MA. Douglas M L. Loveland K A. Sutherland GR. Antalis TM. Testisin, a newhuman serine proteinase expressed by premeiotic testicular germ cellsand lost in testicular germ cell tumors. Cancer Research.59(13):3199-205, 1999.) Prostasin serine protease is an acidic protein(pI 4.5-4.8) of approximately 40 kDa in molecular mess (Yu JX. Chao L.Chao J. Prostasin is a novel human serine proteinase from seminal fluid.Purification, tissue distribution, and localization in prostate gland.Journal of Biological Chemistry. 269(29):18843-8, 1994). It ispredominantly made in the prostate gland (˜146 ng/mg protein), withlesser amounts (2-6 ng/mg protein) also found in the bronchi, colon,kidney, liver, lung, pancreas, and the salivary glands (Yu JX. Chao L.Chao J. Prostasin is a novel human serine proteinase from seminal fluid.Purification, tissue distribution, and localization in prostate gland.Journal of Biological Chemistry. 269(29):18843-8, 1994). Prostasin issecreted in the prostatic fluid, and can be detected in the semen (˜9μg/ml). Prostasin expression is localized to the epithelial cells ofhuman prostate gland by in situ hybridization histochemistry using anantibody or an anti-sense RNA probe (Yu J X. Chao L. Chao J. Prostasinis a novel human serine proteinase from seminal fluid. Purification,tissue distribution, and localization in prostate gland. Journal ofBiological Chemistry. 269(29):18843-8, 1994; (Yu JX. Chao L. Chao J.Molecular cloning, tissue-specific expression, and cellular localizationof human prostasin mRNA. Journal of Biological Chemistry.270(22):13483-9, 1995). Molecular cloning of a full-length humanprostasin cDNA revealed that its predicted amino acid residue sequencecontains a carboxyl-terminal hydrophobic region that can potentiallyanchor the protein on the membrane (Yu JXChao L. Chao J. Molecularcloning, tissue-specific expression, and cellular localization of humanprostasin mRNA. Journal of Biological Chemistry. 270(22):13483-9, 1995).At the amino acid level, prostasin is similar to plasma kallikrein,coagulation factor XI, hepsin, plasminogen, acrosin, prostase, and, inparticular, testisin (sharing 44% sequence identity) [Nelson PS. Gan L.Ferguson C. Moss P. Gelinas R. Hood L. Wang K. Molecular cloning andcharacterization of prostase, an androgen-regulated serine protease withprostate-restricted expression. Proceedings of the National Academy ofSciences of the United States of America 96(6):3114-9, 1999; Hooper J D.Nicol D L. Dickinson J L. Eyre H J. Scarman A L. Normyle J F. Stuttgen MA. Douglas M L. Loveland K A. Sutherland G R. Antalis T M. Testisin, anew human serine proteinase expressed by premeiotic testicular germcells and lost in testicular germ cell tumors. Cancer Research.59(13):3199-205, 1999; Yu JX. Chao L. Chao J. Molecular cloning,tissue-specific expression, and cellular localization of human prostasinmRNA. Journal of Biological sodium channel-activating protease (CAP1)was shown to be highly homologous to human prostasin as well (sharing53% sequence identity at the amino acid level) (Vallet V. Chraibi A.Gaeggeler H P. Horisberger J D. Rossier B C. An epithelial serineprotease activates the amiloride-sensitive sodium channel. Nature.389(6651):607-10, 1997). Prostasin is encoded by a single-copied gene,which is located on human chromosome 16pl 1.2 (Yu JX. Chao L. Ward D C.Chao J. Structure and chromosomal localization of the human prostasin(PRSS8) gene. Genomics. 32(3):334-40,1996). The secreted prostasincleaves synthetic substrates in vitro preferentially at thecarboxyl-terminal side of Arg residue, and is thus considered atrypsin-like serine protease (Yu JX. Chao L. Chao J. Prostasin is anovel human serine proteinase from seminal fluid. Purification, tissuedistribution, and localization in prostate gland. Journal of BiologicalChemistry. 269(29): 18843-8, 1994). The physiological function ofprostasin, however, has remained unknown. By comparing gene expressionof normal tissues, pre-invasive cancer, and invasive cancer, it would behighly advantageous to discover molecular markers that display adifferential expression pattern between the pre-invasive and theinvasive phenotypes and use these markers for a more precise diagnosisand prognosis of prostate and/or breast cancers. With the enhancedprecision in diagnosis and prognosis, treatment options for patientswith DCIS could then be stratified. Those with low risks of developinginvasive breast cancer will have a higher confidence in choosingbreast-conserving options, while those at high risks will ponder moreaggressive options with the necessary follow-up treatments. Similarly,those males with low risks of developing invasive prostate cancer willhave a higher confidence in choosing prostate-conserving options, whilethose at high risks will ponder more aggressive options with thenecessary follow-up treatments.

SUMMARY OF THE INVENTION

The first objective of the present invention is to reduce deficienciesin the prior art with specific regard to differential diagnosis ofinvasive prostate and breast cancers and to treatment of invasive andmetastatic prostate and breast cancers.

The second objective of the present invention is to provide a new markerfor prostate and breast cancer.

The third objective of the invention is to provide as a drug to patientswith carcinoma of the prostate via delivery of a functional prostasingene.

The fourth objective of the invention is to provide as a drug topatients with carcinoma of the prostate via delivery of a functionalprostasin cDNA.

The fifth objective of the invention is to provide as a drug to patientswith carcinoma of the breast via delivery of a functional prostasingene.

The sixth objective of the invention is to provide as a drug to patientswith carcinoma of the breast via delivery of a functional prostasincDNA.

This invention identifies prostasin serine protease as a potentialmarker, and as a tumor invasion suppressor for prostate and breastcancers and thus provides methods (a), (b), and (c) of determininginvasiveness levels of human carcinomas:

-   -   (a) using prostasin protein levels, comprising the steps of:        sampling a human carcinoma tissue; determining prostasin protein        levels in the human carcinoma tissue; preferably by applying an        immunological reagent-antibody to said tissue wherein the        reagent-antibody becomes bound to prostasin protein in said        tissue, and determining invasiveness of the human carcinoma        tissue based on the prostasin protein levels:. or    -   (b) using prostasin mRNA levels, comprising the steps of:        sampling a human carcinoma tissue; determining prostasin mRNA        levels in the human carcinoma tissue preferably by applying        prostasin-specific anti-sense RNA probes in an in situ        hybridization to determine the prostasin mRNA levels in the        separated human carcinoma tissue the determination of the        prostasin mRNA levels in the separated human carcinoma sample        tissue; to make possible and determining invasiveness of the        human carcinoma tissue based on the prostasin mRNA levels; or    -   (c) using prostasin gene promoter DNA methylation levels,        comprising the steps of: sampling a human carcinoma tissue;        determining prostasin gene promoter DNA methylation levels in        the human carcinoma tissue, preferably by applying        prostasin-promoter-specific oligonucleotide primers in a PCR to        determine the prostasin gene promoter DNA methylation levels in        the sampled human carcinoma tissue and determining invasiveness        of the human carcinoma tissue based on the prostasin gene        promoter DNA methylation levels,    -   as well as a method of treating invasive human carcinomas        comprising the steps of: incorporating human prostasin nucleic        acid into a selected gene delivery vector nucleic acid to form a        recombinant nucleic acid; preferably wherein the nucleic acid        includes a gene or cDNA delivering the recombinant nucleic acid        into a human carcinoma; and reducing invasiveness of the human        carcinoma.

Further objects and advantages of this invention will be apparent fromthe following detailed description of a presently preferred embodiment,which is illustrated schematically in the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a diagrammatic representation of the analysis of prostasinindicative of the absence or existence, or of the invasiveness of humanprostate carcinoma.

FIG. 2 is a diagrammatic representation of the analysis of prostasinindicative of the absence or existence, or of the invasiveness of humanmammary carcinoma

FIG. 3 shows human prostasin expression in prostate epithelial cells.

FIG. 4A shows immunohistochemical detection of prostasin protein inbenign human prostate tissues.

FIG. 4B is an enlarged view of the boxed region of FIG. 4A.

FIG. 4C shows immunohistochemistry of benign human prostate tissues withprostasin antibody omitted in the procedures, no epithelial cellsdisplayed any staining.

FIG. 4D is an enlarged view of the boxed region of FIG. 4C.

FIG. 4E shows immunohistochemistry of human prostate tumor withprostasin antibody omitted in the procedures, no epithelial cellsdisplayed any staining.

FIG. 4F shows immunohistochemical detection of prostasin protein inbenign human prostate tissue surrounded by prostate carcinomas.

FIG. 4G shows immunohistochemical detection of prostasin protein inGleason grade 2 prostate tumor.

FIG. 4H is an enlarged view of the boxed region of FIG. 4G.

FIG. 4I shows immunohistochemical detection of prostasin protein inGleason grade 3 prostate tumor.

FIG. 4J is an enlarged view of the boxed region of FIG. 41.

FIG. 4K shows immunohistochemical detection of prostasin protein inGleason grade 4 prostate tumor.

FIG. 4L is an enlarged view of the boxed region of FIG. 4K.

FIG. 5 shows human prostasin expression in human breast cancer celllines.

FIG. 6 shows promoter hypermethylation of the human prostasin gene inhuman prostate and breast cancer cell lines.

FIG. 7 shows prostasin protein expression and in vitro invasiveproperties of the DU-145 and the PC-3 transfectants.

FIG. 8 shows prostasin protein expression and in vitro invasiveproperties of the MDA-MB-231 and the MDA-MB-435s transfectants.

FIG. 9 a Is a schematic illustration of the promoter-exon region of theprostasin gene.

FIG. 9 b shows a genomic Southern blot analysis of DNA from NHMEC andhuman breast cancer cell lines.

FIG. 9 c shows a genomic Southern blot analysis of MDA-MB-231 andMDA-MB-435s cells following demethylation.

FIG. 10 shows DNA sequence and the deduced amino acid sequence of thePRSS8 (prostasin) gene.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Before explaining the disclosed embodiment of the present invention indetail it is to be understood that the invention is not limited in itsapplication to the details of the particular arrangement shown since theinvention is capable of other embodiments. Also, the terminology usedherein is for the purpose of description and not of limitation.

Using RT-PCR and western blot analyses, prostasin protein and mRNAexpression were found in normal human prostate epithelial cells and thehuman prostate cancer cell line LNCaP, but discovered not present in thehighly invasive human prostate cancer cell lines DU-145 and PC-3.Immunohistochemistry studies of human prostate cancer specimens revealeda down-regulation of prostasin in high-grade tumors.

Using RT-PCR and western blot analyses, prostasin protein and mRNAexpression were found in a non-invasive human breast cancer cell line,MCF-7, while invasive human breast cancer cell lines MDA-MB-231 andMDA-MB-435s were also discovered not to express either the prostasinprotein or the mRNA. A non-invasive human breast cancer cell line,MDA-MB-453, was shown to express prostasin mRNA but not prostasinprotein. Examination of the prostasin gene promoter in the humanprostate and breast cancer cell lines by Southern blot analysis revealedheterogeneous methylation of the promoter in DU-145, PC-3 and MDA-MB-453cells, and homogeneous methylation of the promoter in MDA-MB-231 andMDA-MB-435s cells. The prostasin gene promoter in normal human prostateepithelial cells, the LNCaP and the prostasin gene promoter in the humanprostate and breast cancer cell lines by Southern blot analysis revealedheterogeneous methylation of the promoter in DU-145, PC-3 and MDA-MB-453cells, and homogeneous methylation of the promoter in MDA-MB-231 andMDA-MB-435s cells. The prostasin gene promoter in normal human prostateepithelial cells, the LNCaP and the MCF-7 cells was shown to beunmethylated. Transfection of DU-145 and PC-3 cells with a full-lengthhuman prostasin cDNA restored prostasin expression and reduced the invitro invasiveness by 68% and 42%, respectively. Transfection ofMDA-MB-231 and MDA-MB-435s cells with a full-length human prostasin cDNArestored prostasin expression and reduced the in vitro invasiveness by50% for either cell line. Cell proliferation was unaffected byre-expression of prostasin. Our data indicate that prostasin isimplicated in normal prostate biology and its down-regulation inprostate cancer, and its absence in invasive prostate and breast cancercell lines indicates increased invasiveness. Our results also indicatethat delivering a functional human prostasin gene to invasive prostateand breast cancers can reduce the invasiveness.

For a facile understanding of the invention embodied herein, referenceshould now be made to FIGS. 1 and 2. FIG. 1 sets forth a schematic fordetermining if a male has prostate cancer. Human blood 10 is taken fromthe male and analyzed for the presence of prostasin serine protease 14.If NO 12 prostasin is found, there is probably little or no cancer. Ifthere is a presence YES 16 of prostasin serine protease 14, there isprostate cancer. If a biopsy of the prostate gland 20 is analyzed forthe presence of a normal amount YES 22 of prostasin serine protease 14,there is no cancer or there is non-invasive cancer. If there is NO 24 orReduced 26 amount of prostasin serine protease 14, there is invasivecancer.

FIG. 2 sets forth a schematic for determining if a female has breastcancer. Human blood 100 is taken from the female and analyzed for thepresence of prostasin serine protease 140. If NO 120 prostasin is found,there is prospectively no cancer. If there is a presence YES 160 ofprostasin serine protease 140, there is breast cancer. If a biopsy ofthe breast 200 is analyzed for the presence of YES 220 of prostasinserine protease 140, there is no invasive cancer but may be non-invasivecancer. If there is NO 240 or Reduced 260 amount of prostasin serineprotease 140, there is invasive cancer.

The levels of prostasin protein in the epithelial cells of the humanprostate gland can be used as a diagnostic marker for the potentialinvasiveness of prostate tumors. The supporting evidence came from ourfindings that two invasive human prostate cancer cell lines DU-145 andPC-3 do not express prostasin while normal prostate epithelial cells anda non-invasive prostate cancer cell line LNCaP express both theprostasin mRNA and protein.

Refer now to FIG. 3 which shows human prostasin expression in prostateepithelial cells. By means of western blot analysis (upper panel),prostasin (as a 40-kDa band) was detected in normal human prostateepithelial cells (CC-2555) and the LNCaP cells, but not in the DU-145 orPC-3 cells. An equal amount of total protein (100 μg) was loaded foreach sample. At the mRNA level, human prostasin mRNA (via a 232-bpamplified DNA band) was detected in normal prostate epithelial cells(CC-2555) and the LNCaP cells, but not in the DU145 or PC-3 cells asanalyzed by RT-PCR/Southern blot hybridization (middle panel).Co-amplification of a 556-bp human β-actin message (as shown in the gelphotograph in the lower panel) confirmed the quality and the quantity ofthe RNA applied in each RT-PCR.

Expression of prostasin protein is reduced in high-grade human prostatetumor. Prostatectomy specimens from 39 patients (128 sections) weresubjected to immunohistochemistry using a prostasin-specific antibody.Overall, in non-tumor or benign prostate epithelia, 89.0% of theexamined areas demonstrated positive staining for prostasin protein and11.0% were considered negative (based on the scoring system used forHercepTest™, DAKO Corporation, Carpinteria, CA). In all tumor specimensthat were examined, prostasin was detected in 93.3% of the low Gleasongrade areas (≦grade 2), 44.4% of Gleason grade 3 areas, 21.1% of Gleasongrade 4 areas, but not in Gleason grade 5 areas (data summarized inTable 1). The mean prostasin immunostaining score was foundsignificantly decreased in high-grade prostate tumors as compared tonon-tumor areas (ANOVA, p<0.0001).

Representative staining images of non-tumor (benign) areas and prostatetumor areas are shown in FIGS. 4 a-4 l, which providesimmunohistochemical detection of prostasin protein in tissues.Paraffin-embedded human prostate sections were stained for prostasinprotein expression evaluation using a specific antibody as described (YuJX. Chao L. Chao J. Prostasin is a novel human serine proteinase fromseminal fluid. Purification, tissue distribution, and localization inprostate gland. Journal of Biological Chemistry. 269(29):18843-8, 1994).Prostasin positive staining (brown color) was detected in the cytoplasmand apical membrane in non-tumor or benign epithelial cells.

The prostasin protein was detected in the cytoplasm and on the plasmamembrane (apical) of benign epithelial cells lining the secretory lumenas well as in the secretion inside the lumen (FIGS. 4A and 4B, score 3,or +++), confirming the results of (Yu JX. Chao L. Chao J. Prostasin isa novel human serine proteinase from seminal fluid. Purification, tissuedistribution, and localization in prostate gland. Journal of BiologicalChemistry. 269(29):18843-8, 1994). When a pre-immune rabbit serum wasused in place of the prostasin antiserum, no staining was observed ineither the non-tumor epithelia (FIGS. 4C and 4D) or tumor epithelia(FIG. 4E). Tumor epithelia displayed various degrees of prostasinimmunostaining as shown in FIGS. 4F-4L. In Gleason grade 1-2 tumors,moderate prostasin staining is seen in the cytoplasm and on the plasmamembrane of some epithelial cells, as well as in the secretion in thelumen (FIGS. 4G and 4H, score 2, or ++). In Gleason grade 3 tumors, alesser number of epithelial cells displayed the moderate level prostasinstaining (FIGS. 4I and 4J). In Gleason grade 4 tumors, most epithelialcells did not show any prostasin staining, while some prostasin stainingcan be seen in rare, sporadic tumor cells (FIGS. 4K and 4L, as indicatedby the arrow, score 0). Genetically, prostate tumors are heterogeneousand multi-focal in nature, in that one patient's gross-anatomy tumorcomes from multiple initial lesions which are caused by differentinitial transformation events and progress to different stages bydifferent ensuing transformations (Isaacs J T. Bova G S. ProstateCancer. In The Genetic Basis of Human Cancer (Eds. Vogelstein B andKinzler KW), pp653-60, McGraw-Hill Health Professions Division, 1998).The Gleason grading, when used as a percentage of each cancer occupiedby Gleason grade ⅘ areas, is independently associated with prostatecancer progression (Stamey T A, McNeal J E, Yemoto C M, Sigal B M,Johnstone I M. Biological determinants of cancer progression in men withprostate cancer. JAMA 281:1395-1400, 1999). We found a significantdecrease of prostasin expression in the high-grade, i.e., the moreprogressively transformed tumors.

As earlier indicated, FIG. 5 shows human prostasin expression in humanbreast cancer cell lines. By means of western blot analysis (upperpanel), prostasin (as a 40-kDa band) was detected in MCF-7 cells, butnot in MDA-MB453, MDA-MB-231, or MDA-MB-435s cells. An equal amount oftotal protein (100 μg) was loaded for each sample. At the mRNA level,human prostasin mRNA (via a 232-bp amplified DNA band) was detected inMCF-7 and MDA-MB453 cells, but not in the MDA-MB-231 or MDA-MB-435scells as analyzed by RT-PCR/Southern blot hybridization (lower panel).

Prostasin mRNA expression is seen absent in two invasive human breastcancer cell lines while two non-invasive breast cancer cell linesexpress prostasin mRNA or protein. Analysis of prostasin expression inhuman breast cancer cell lines showed that the non-invasive MCF-7 andMDA-MB-453 cells express the prostasin mRNA while the highly invasiveMDA-MB-231 and MDA-MB-435s cells do not express the prostasin mRNA (seeFIG. 5). Expression of prostasin mRNA in normal human breast can bedemonstrated by the presence of two GenBank™ normal human breast ESTsequences coding for prostasin (Accession numbers R48653, and R48557).The MCF-7 cells also express the prostasin protein as determined bywestern blot analysis (again see FIG. 5). Prostasin down-regulation inprostate or breast cancer can be caused by promoter methylation orgene-specific mutation. Prostasin expression decreases with increasingprostate cancer grade and is absent in invasive prostate and breastcancer cell lines. The chromosomal locus where the human prostasin geneis, 16p11.2, however, is not known to be an LOH hot-spot in prostatecancer or in breast cancer. Epigenetic events (such as DNA methylation)may be an alternative mechanism of loss of expression for tumorsuppressors or invasion suppressors. Refer now to FIG. 6 which showspromoter hypermethylation of the human prostasin gene in human prostateand breast cancer cell lines. Genomic DNA (5 μg) from the various celllines (as indicated in the figure) were digested with the followingrestriction enzyme combinations, Xho I/BamH I (X/B, flanking cuts of themethylation-sensitive site), Xho I/BamH I/Msp I (X/B/M), or Xho I/BamHI/Hpa II (X/B/H). The digests were resolved in a 0.8% agarose gel andtransferred to an Immobilon-N membrane for hybridization with anick-translated prostasin promoter probe (bases 703-1,649 of theprostasin gene sequence U33446). The probe detects a promoter fragmentof 1,275 bp, which is cut by the methylation-insensitive enzyme Msp I toyield a 951-bp fragment for all DNA samples. The methylation-sensitiveisoschizomer Hpa II yields the 951-bp fragment in the CC-2555 (normalprostate epithelial cells), the LNCaP, and the MCF-7 samples, indicatingthe hypomethylated or unmethylated state of the prostasin promoter inthese cells. For DU-145, PC-3, and MDA-MB453 DNA, both the 951-bp andthe 1,275-bp fragments are generated in the methylation-sensitivedigestion, suggesting incomplete methylation (one of two or morechromosomes) or clonal methylation in a sub-population of cells. For theMDA-MB-231 and MDA-MB-435, however, the Hpa II digestion did not yieldthe 951-bp but rather gave the 1,275-bp fragment. This homogeneousmethylation pattern indicates that the Msp I/Hpa II site, at location-96(relative to the transcription initiation site) of the prostasinpromoter, is methylated (hypermethylated) in these DNA samples.

Signal intensity variation may be attributed to aneuploidy. (In additionto the Hpa II site at base number 1326/−96 shown in FIG. 6, example 1and 2 are provided, more extensively detailing the other prostasin genepromoter methylated sites and experimental methods for determining them.Such sites have been located at bp 615/−807, 1,102/−320, 1,156/−266,1,326/−96 and 1,445/+24. Nomenclature used in describing the nucleicacid sequences of the prostasin gene are described by Yu, et al inStructure and Chromosomal localization of the human prostasis (PRSS8)gene. Genomics 1996:32 pp. 334-40); page 336 of the Yu, et al referenceis incorporated as FIG. 10 in the description of the present invention.

An examination of the prostasin gene promoter region for DNA methylationdifferences among human prostate and breast cancer cell lines has beenmade (see FIG. 6). We found that cells that express prostasin, normalprostate epithelial, LNCaP, and MCF-7, are unmethylated in the prostasinpromoter while MDA-MB-453 showed heterogeneous prostasin promotermethylation. For cells that do not express prostasin, DU-145 and PC-3showed heterogeneous prostasin promoter methylation while MDA-MB-231 andMDA-MB-435 showed homogeneous hypermethylation in the promoter region ofthe prostasin gene.

Two human prostate cancer cell lines that do not express prostasin, thehighly invasive DU-145 and PC-3, show heterogeneous methylation in thepromoter region of the prostasin gene. The result suggests that at leastone of the two (or more) chromosome 16's of these cell lines ismethylated at the prostasin gene locus. The prostasin gene on theunmethylated chromosome may contain mutations that silenced theexpression. An alternative explanation for the heterogeneous methylationpattern is that the methylation occurs in clonal cell populations,however, the lack of detectable prostasin mRNA in our RT-PCR-Southernblot analysis in the DU-145 and PC-3 cells argues against thispossibility.

The significance of the finding on prostasin gene promoterhypermethylation in prostate or breast cancer is that the measurement ofprostasin down-regulation as a cancer marker may be achieved by using abinary assay (yes-or-no), instead of a gradually decreasing quantity inthe immunohistochemistry assay (which is quite arbitrary).

Re-expression of human prostasin protein in invasive human prostate andbreast cancer cells reduces invasiveness in vitro. At this point,reference should be made to FIG. 7 which shows the prostasin proteinexpression and in vitro invasive properties of the DU-145 and the PC-3transfectants. DU-145 or PC-3 cells transfected with either a vector DNA(labeled as “vector”) or a prostasin cDNA construct (labeled as“prostasin”) were analyzed by a western blot analysis using aprostasin-specific antibody (upper panel) or subjected to an in vitroMatrigel chemoinvasion assay (lower panel) as described in (Liu DF.Rabbani SA. Induction of urinary plasmiinogen activator by retinoic acidresults in increased invasiveness of human prostate cancer cells PC-3.Prostate. 27(5):269-76, 1995). The expressed human prostasin protein (a40-kDa band) was detected in the prostasin cDNA-transfected DU-145 orPC-3 cells, but not in the vector-transfected cells. In the Matrigelchemoinvasion assay, the vector-transfected cells are expressed as being100% invasive (solid bar), the open bar represents the relativeinvasiveness of the human prostasin cDNA-transfected cells. The datawere analyzed by a Student t-test using the StatView software (AbacusConcepts, Inc., Berkeley, Calif.).

It can be seen that Polyclonal DU-145 and PC-3 cells transfected withthe human prostasin cDNA (designated DU-145/Pro, and PC-3/Pro,respectively) were confirmed to express the human prostasin protein, asshown in the western blot analysis of the cell lysate (FIG. 7, upperpanel). The vector-transfected cells, designated DU-145/Vector orPC-3/Vector, respectively, were used as negative control in the westernblot. A further examination of the DU-145/Pro and the PC-3/Pro cells byimmunocytochemistry confirmed that 100% of the cells expressed theprostasin protein (data not shown). In in vitro Matrigel chemoinvasionassays (FIG. 7, lower panel), the invasiveness of DU-145/Pro cells wasdetermined to be at 32% of that of DU-145Nector cells (or, the reductionof invasiveness was at 68%). The invasiveness of PC-31Pro cells wasdetermined to be at 58% of that of PC-3Nector cells (or, the reductionof invasiveness was at 42%). We performed in vitro cell proliferationassays on DU-145/Pro vs. DU-145/Vector cells, and on PC-3/Pro vs.PC-3/Vector cells, but did not observe any difference between the growthrates of the prostasin cDNA-transfected or the vector-transfected cellsover an 8-day period (data not shown).

Forced re-expression of human prostasin in two invasive human breastcancer cell lines reduced invasiveness. A full-length human prostasincDNA under the control of an RSV promoter was transfected into theinvasive breast cancer MDA-MB-231 and MDA-MB-435 cells. Reference shouldbe made to FIG. 8 which shows prostasin protein expression and in vitroinvasive properties of the MDA-MB-231 and the MDA-MB-435s transfectants.MDA-MB-231 and MDA-MB-435s cells transfected with either a vector DNA(labeled as “vector”) or a prostasin cDNA construct (labeled as“prostasin”) were analyzed by a western blot analysis using aprostasin-specific antibody (upper panel) or subjected to an in vitroMatrigel chemoinvasion assay (lower panel) as referenced in (Liu DF.Rabbani SA. Induction of urinary plasminogen activator by retinoic acidresults in increased invasiveness of human prostate cancer cells PC-3.Prostate. 27(5):269-76, 1995). The expressed human prostasin protein (a40-kDa band) was detected in the prostasin cDNA-transfected MDA-MB-231and MDA-MB-435s cells, but not in the vector-transfected cells. In theMatrigel chemoinvasion assay, the vector-transfected cells are expressedas being 100% invasive (solid bar), the open bar represents the relativeinvasiveness of the human prostasin cDNA-transfected cells. The datawere analyzed by a Student t-test using the StatView software (AbacusConcepts, Inc., Berkeley, Calif.).

Stable, polyclonal, episomal transfectants were obtained and theexpression of human prostasin protein was confirmed by western blotanalysis (FIG. 8, upper panel). In in vitro Matrigel chemoinvasionassays, the invasiveness of either cell lines expressing human prostasinwas reduced by 50% as compared to the vector-transfected controls (FIG.8, lower panel).

Taken together, the foregoing evidences linking prostasin levelreduction or protasin absence to the invasiveness of prostate and breastcancer cell lines, or linking prostasin expression to reducedinvasiveness. The evidence qualifies prostasin as an invasionsuppressor, which thus is a marker for diagnosis of invasiveness ofprostate and breast cancers, or as a therapeutic agent to treat invasiveprostate and breast cancers.

Pathological grading by the Gleason system is performed after eithersurgery or biopsy, both highly invasive procedures, while blood testssuch as that for the PSA prostate cancer marker can offer the hope ofaccurate diagnosis and prognosis without the harm of an invasiveprocedure. In practice, however, single markers suffer from an intrinsiclimitation that the “positive” identifications are not always confirmedfor the “diagnosed” disease. Biopsy is still required for a truepositive identification of prostate cancer even in the case of theapplication of the PSA prostate cancer marker (Catalona W J. Partin A W.Slawin K M. Brawer M K. Flanigan R C. Patel A. Richie J P. deKemion J B.Walsh P C. Scardino P T. Lange P H. Subong E N. Parson R E. Gasior G H.Loveland K G. Southwick P C. Use of the percentage of freeprostate-specific antigen to enhance differentiation of prostate cancerfrom benign prostatic disease: a prospective multicenter clinical trial[see comments]. JAMA. 279(19):1542-7, 1998). As stated above, it hasbeen demonstrated in principle that many markers used in a multivariateapproach may provide a highly accurate diagnosis (Greene G F. Kitadai Y.Pettaway Calif. von Eschenbach A C. Bucana C D. Fidler I J. Correlationof metastasis-related gene expression with metastatic potential in humanprostate carcinoma cells implanted in nude mice using an in situmessenger RNA hybridization technique. American Journal of Pathology.150(5):1571-82, 1997). From the standpoint of prostate cancer genetics,the multivariate approach is well supported by our currentunderstanding. A serine protease structurally and genetically related tothe PSA, the hK2 (human glandular kallikrein 2) has shown some promiseof joining the list of markers applicable for prostate cancer diagnosis(Saedi M S. Hill T M. Kuus-Reichel K. Kumar A. Payne J. Mikolajczyk S D.Wolfert R L. Rittenhouse H G. The precursor form of the human kallikrein2, a kallikrein homologous to prostate-specific antigen, is present inhuman sera and is increased in prostate cancer and benign prostatichyperplasia. Clinical Chemistry. 44(10):2115-9, 1998). Structurally andin prostate gland biology, prostasin shares many common characteristicswith both PSA and hK2, as being a secreted serine protease made in largeabundance in prostate epithelial cells (Yu JX. Chao L. Chao J. Prostasinis a novel human serine proteinase from seminal fluid. Purification,tissue distribution, and localization in prostate gland. Journal ofBiological Chemistry. 269(29):18843-8, 1994; Yu JX. Chao L. Chao J.Molecular cloning, tissue-specific expression, and cellular localizationof human prostasin mRNA. Journal of Biological Chemistry.270(22):13483-9, 1995). While high-grade prostate cancer cells produceless PSA protein than normal prostate cells or low-grade prostate cancercells (Hakalahti L. Vihko P. Henttu P. Autio-Harmainen H. Soini Y. VihkoR. Evaluation of PAP and PSA gene expression in prostatic hyperplasiaand prostatic carcinoma using northern-blot analyses, in situhybridization and immunohistochemical stainings with monoclonal andbispecific antibodies. International Journal of Cancer. 55(4):590-7, 1;Sakai H. Yogi Y. Minami Y. Yushita Y. Kanetake H. Saito Y. Prostatespecific antigen and prostatic acid phosphatase immunoreactivity asprognostic; Sakai H. Yogi Y. Minami Y. Yushita Y. Kanetake H. Saito Y.Prostate specific antigen and prostatic acid phosphataseimmunoreactivity as prognostic), the serum PSA levels in prostate cancerpatients increase due to tissue damage caused by invasive cancer(Rittenhouse H G. Finlay J A. Mikolajczyk S D. Partin A W. HumanKallikrein 2 (hK2) and prostate-specific antigen (PSA): two closelyrelated, but distinct, kallikreins in the prostate. [Review] [457 refs]Critical Reviews in Clinical Laboratory Sciences. 35(4):275-368, 1998).By comparison, we also believe prostasin is in the circulation ofprostate cancer patients. As a result, a blood test for the circulatingprostasin to indicate the presence and/or the stage of prostate cancerwould be highly useful. (as illustrated in FIG. 1). In principal, thefeasibility of a blood test based on prostasin detection to indicatecancer has been demonstrated by Berteau et al. (1999). These authors(Berteau P. Laribi Eschwege P. Lebars I. Dumas F. Benoit G. Loric S.Prostasin mRNA to detect prostate cells in blood of cancer patients.Clinical and Chemical Laboratory Medicine 37 (SS): SI 19, 1999),demonstrated a highly promising potential of using prostasin as a markerto detect circulating prostate epithelial cells, a sign of prostatetissue damage caused by invasive prostate cancer leading to thedissemination of prostate epithelial cells into the circulation. It isexpected that a blood test for circulating prostasin to indicate thepresence and/or the stage of breast cancer would be highly useful. (asillustrated in FIG. 2).

In summary of the invention, it has been taught herein that: proteinprostasin as well as its MRA levels and its gene promoter DNAmethylation levels can be used to determine the invasiveness level ofhuman carcinomas; and, provide a method of treating invasive humancarcinomas by delivery thereto of a recombinant nucleic acid formed by ahuman prostasin nucleic acid incorporated into a selected gene deliveryvector. Those teachings are repeated for emphasis in the following:

-   -   1. Immunohistochemistry studies of human prostate cancer        specimens revealed a down-regulation of prostasin in high-grade        tumors.

2. Using RT-PCR and western blot analyses, prostasin protein and mRNAexpression were found in a non-invasive human breast cancer cell line,MCF-7, while invasive human breast cancer cell lines MDA-MB-231 andMDA-MB-435s were found not to express either the prostasin protein orthe mRNA. A non-invasive human breast cancer cell line, MDA-MB-453, wasshown to express prostasin mRNA but not prostasin protein: and,

-   -   3. Examination of the prostasin gene promoter in the human        prostate and breast cancer cell lines by Southern blot analysis        revealed heterogeneous methylation of the promoter in DU-145,        PC-3 and MDA-MB453 cells, and homogeneous methylation of the        promoter in MDA-MB-231 and MDA-MB-435s cells. The prostasin gene        promoter in normal human prostate epithelial cells, the LNCAP        and the MCF-7 cells was shown to be unmethylated. Transfection        of DU-145 and PC-3 cells with a full-length human prostasin cDNA        restored prostasin expression and reduced the in vitro        invasiveness by 68% and 42%, respectively. Transfection of        MDA-MB-231 and MDA-MB-435s cells with a full-length human        prostasin cDNA restored prostasin expression and reduced the in        vitro invasiveness by 50% for either cell line.

The preferred methods of separating sampled human carcinoma tissue fromneighboring normal tissues is by laser capture micro-dissection.

The following examples are provided for the purpose of illustration andnot limitation:

Example 1

Prostasin Promoter DNA Methylation

Cell Culture Maintenance

All cell culture media, sera and supplements were purchased from LifeTechnologies (Gaithersburg, Md.) except for those otherwise noted

A normal human mammary epithelial cell primary culture (catalog numberCC-255) was obtained from Clonetics (San Diego, Calif.) and maintainedin the mammary epithelial basal medium (supplemented with bovinepituitary extract, recombinant human epidermal growth factor, bovineinsulin and hydrocortisone) according to the suppliers protocols. TheCulture was kept at 37° C. with 5% CO₂ and used for experiments at the9^(th) overall passage.

Human breast carcinoma cell lines MCF-7, MDA-MB_(—)453, MDA-MB-231 andMDA_MB-435 were obtained from the American Type Culture Collection(ATCC, Manassas, Va.). The MCF-7 cells were maintained in DMEMsupplemented with 10% FBS and kept at 37 for experiments at the 9^(th)overall passage.

Human breast carcinoma cell lines MCF-7, MDA-MB_(—)453, MDA-MB-231 andMDA_MB-435 were obtained from the American Type Culture Collection(ATCC, Manassas, Va.). The MCF-7 cells were maintained in DMEMsupplemented with 10% FBS and kept at 37° C. with 5% CO₂. The MDA-MB-231and MDA-MB-453 cells were maintained in Lebovitz-15 (L-15) mediumsupplemented with 10% FBS and kept at 37° C. without CO₂. The MDA-MB-435cells were maintained in L-15 medium supplemented with 15% FBS and 10μg/ml bovine insulin and kept at 37° C. without CO₂.

Transfection Of Cell Lines With Plasmid DNA And Selection Of StableTransfectants

Construction of a plasmid containing a full-length human prostasin cDNAunder the control of a Rous sarcoma virus (RSV) promoter andtransfection of cells were carried out as described in Chen, L M, Hodge,G B, Guarda, L A et al. Down-Regulation of Prostasin Serine Protease, Apotential Invasion Suppressor in Prostate Cancer, Prostrate2001,48:93-103. Briefly, 1,000,000 MDA-MB-231 or MDA-MB-435s cells wereresuspended in 0.3 ml of the culture medium and mixed with 50 μg ofplasmid DNA dissolved in 0.1 ml of sterile distilled cuvette and pulsedat 200 volts, 1,600 μF, 72 ohms and 500 V/capacitance setting on aBTX-600 Eleciro-cell manipulator (Genetronics, San Diego, Calif.).Selection of transfectants was carried out in the presence of 800 μg/mlG418 (final concentration) until colonies appeared. (5-7 days) Colonies(≈200 in number) were then dispersed via trypsinization and maintainedin G418 containing culture medium without colony-isolation. Cellstransfected with the human prostasin cDNA construct were assayed inWestern blot analysis for expression of the prostasin protein, usingvector-transfected cells as controls.

RNA Preparation And Analysis By RT-PCR(Southern Blot

Cells grown to 80% confluence (in 60 nm tissue culture dish) were lyseddirectly with 1 ml of the Trizol Reagent (life Technologies) and RNA wasisolated according to the manufacturer's protocol. The humanprostasin-specific RT-PCR/Southern blot analysis was performed asdescribed in Yu, J X, and Chao,J Molecular Cloning, tissue-specificexpression and cellular localization of human prostasin mRNA. J BiolChem 1995; 270:12483-4. One microgram of total RNA was used in theRT-PCR with 2 human prostasin gene-specific oligonucleotide primers, anda Southern blot of the resolved RT-PCR samples was probed with a thirdprostasin gene-specific oligonucleotide, detecting an amplified 232 bpfragment. A co-amplification of the β-actin mRNA for control of RNAquality and quantity was performed as described in Chen Hodge andGuarda, et al.

Western Blot Analysis

Cells grown to 80-100% confluence were washed 3 times in 1×PBS (pH 7.4)and then lysed in RIPA buffer (1×PBS, pH 7.4, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS). The total lysate were centrifuged at 14,000 rpmfor 30 minutes at 4° C. to remove the pellet. Protein concentration wasdetermined using a DC (detergent-compatible) protein assay kit (Bio-Rad,Hercules, Calif.). The samples were then subjected to SDS-PAGE followedby Western blot analysis using a prostasin-specific antibody. Briefly,cell lysate were resolved in 10% gels under reducing conditions beforeelectrotransfer to nitrocellulose (NC) membranes (Fisher Scientific,Pittsburgh, Pa.). Upon complete protein transfer, the NC membranes wereblocked in BLOTTO (5% nonfat milk made with Tris-buffered saline/0.1%Tween-20, pH 7.6), incubated with the prostasin-specific antibodydiluted at a ratio of 1:2000 in BLOTTO, followed by an incubation withthe secondary antibody, goat antirabbit IgG conjugated to HRP(horseradish peroxidase; used at 1:10,000) (Sigma-Aldrich, St. LouisMo.). The bound secondary antibody was detected using enhancedchemi-luminesence (ECL) reagents (Pierce, Rockford, Ill.) according tothe suppliers recommendations. All procedures were performed at roomtemperature, and the membranes were washed between steps.

Matrigel Chemoinvasion Assay

The Matrigel chemoinvasion assay was carried out essentially asdescribed in Chen, L. M., Hodge, G B, Guardia, LA et al. Down-Regulationof prostasin serine protease, a potential invasion suppressor inprostate cancer. Prostate 2001:48:93-103, incorporated herein. Basementmembrane Matrigel stock (10 mg/ml; Collaborative Biochemical, Bedford,Mass.) was thawed overnight on ice in a refrigerator (0-4° C.).Transwell invasion chambers (Coster, Cambridge, Mass.) with 8 μm porepolycarbonate filters (growth area 0.33 cm²) were coated with Matrigel(50 μg/filter, a 1:3 dilution of the stock with chilled serum-freemedium was used for coating). The gels were solidified by incubating thecoated filters at 37° C. for 60 min in a moist chamber before use. Thelower chambers of the Transwell plates were filled with 0.6 mlserum-free medium (1-15), supplemented with 25 μg/ml fibronectin(Sigma-Aldrich). Fifty thousand cells in 0.1 ml serum-free OPTI-MEM Imedium were placed onto each Matrigel-coated filter. The filtercartridge was then inserted into the lower chamber and the assays wereperformed at 37° C. for 24 hours (MDA-MB-435s transfectants) or 72 hour(MDA-MB-231 transfectants). After removing the medium, the filters werewashed 3 times in 1×PBS, fixed at room temperature for 20 min in 4%paraformaldehyde made with 0.1 M sodium phosphate buffer (pH 7.4) andthen washed 3 times with the sodium phosphate buffer. The filters werethen stained with 1% toluidine blue (LabChem, Pittsburgh, Pa.) for 2min. The cells on the Matrigel surface were removed with a Q-tip and allcells on the underside of the filters were counted under a lightmicroscope after mounting the filter on a glass slide. All invasionassays were done in triplicate for at least 3 times.

Genomic Southern Blot Analysis Of Prostasin Promoter Methylation

High molecular weight genomic DNA was isolated from various cell linesas described in Chai, K X, Ward, D C, Chao, J, et al Molecular CloningsSequence Analysis and Chromosomal Localization of the Human ProteaseInhibitor 4 (kallistatin) gene (P14) Genomics 1994; 23:370-8. GenomicDNA frm each cell line was digested with the following restrictionenzyme combinations:Xho I/BamH I (X/B), Xho I/BamH I/Hha (X/B/Hh), XhoI/BamH I/Aci I (X/B/A), Xho I/Bam H I.Bsa I (X/B/Bs),Xho I/Bam H I/MspI(X/B/M) or Xho I/Bam H I/Hpa II(X/B/H) using 10 μg of DNA per digestioncombination. The X/B cutting sites flank the CpG sies be to beinvestigated for differential methylation by the methylation-sensitiverestriction endonucleases. The digests were resolved in a 0.8% agarosegel and analyzed by Southern blot hybridization as described in Chai, KX, Ward, D C, Chao, J et al. A nick translated prostasin promoter probe(bases 703-1,649) of the prostasin gene sequence, Genbank accessionnumber U33446) was used for the hybridization. The base numbering of theprostasin gene sequence was described in Yu et al.

Demethylation of Prostasin Gene Promoter and Reactivation of ProstasinExpression

For demethylation of prostasin gene promoter, MDA-MB-231 and MDA-MB-435scells were seeded in 100 mm dishes at an initial density of 50%confluence and cultured in the presence of 500 nM 5-aza-2′-deoxycytidine(5-aza-2′dC; Sigma-Aldrich) for 8 days with renewal of medium and5-aza-2′-dC performed at 2 day intervals. Genomic DNA was then isolatedfor Southern blot analysis as described. For reactivation of prostasinexpression, MDA-MB-231 and MDA-MB-435s cells were seeded in 60 mm dishesat 80% confluence and cultured in the presence of 500 nM 5-aza-2′-dC for24 hr, and were then treated for an additional 24 hr with either 1 μMtrichostatin A (TSA; Sigma Aldrich) or an equal volume of 95% ethanolused to dissolve TSA. For measuring time-dependence of prostatin genereactivation, cells were treated as described above but were harvestedat 6 or 12 hr after the addition of TSA. RNA was isolated forprostasin-specifric RT-PCR/Southern blot analysis as described. TheMCF-7 and the MDA-MB-453 cell ines were subjected tot the same5-aza-2′-dC/TSA treatment and prostasin RT-PCR Southern blot analysisprocedures.

Results

Prostasin Expression In Breast Cancer Lines

Expression of prostasin protein and mRNA in Normal Human MammaryEpithelial Cells (NHMEC) and human breast carcinoma cell lines wasexamined by Western blot analysis using a prostasin-specific antibody orRT-PCR/Southern blot analysis as described. For this experiment 4 breastcarcinoma cell lines based on their differences in invasiveness andmetastatic potential were chosen. The MCF-7 cell line is poorly invasiveto noninvasive, tumorigenic but nonmetastatic. The MDA-MB-453 cells arepoorly tumorigenic and nonmetastatic. The MDA-MB-231 and MDA-MB-435cells are both highly invasive and highly metastatic.

As was shown in FIG. 5 (middle panel) a 232 bp amplified prostasinmessage was detected in total RNA of NHMEC, MCF-7 and MDA-MB-453 but notMDA-MB-231 or MDA-MB-435s. A co-amplification of β-actin message isshown in the bottom panel of FIG. 5 to demonstrate the quality andquality of total RNA used in each RT-PCR. In addition to the expected232 bp prostasin amplification product, a faint bank of retardedmobility was also detected in the Southern blot analysis. This retardedband does not represent a new prostasin message, but rather is asingle-stranded form of the expected prostasin PCR product. In aseparate experiment, we were able to remove this single-stranded form bytreating the PCR products with SI nuclease prior to electrophoresis(data not shown)

Re-expression of recombinant human prostasin in invasive breast cancercells reduced in vitro invasiveness

Following the electroporation and drug selection ≈200 colonies formedfor each transfected cell type. All colonies for each transfected celltype were kept in a mixed culture as polyclonal transfectants for theensuing experiments. Polyclonal MDA-MB-231 and MDA-MB-435s cellstransfected with the human prostasin cDNA were confimred to express therecombinant human prostasin protein, as shown by Western blot analysisof the cell lysate (FIG. 8, top panel) The vector transfected cells wereused as negative control in the Western blot analysis.

In the in vitro Matrigel chemoinvasion assays shown in FIG. 8, bottompanel) transfected MDA-MB-231 or MDA-MB-345s cells expressing therecombinant human prostasin protein showed a significantly reduced levelof invasiveness, at 50% of that of their vector transfected controls,respectively.

Prostasin Gene Promotor Is Hypermethylated In Invasive Breast CancerLines

Examination of the prostasin gene locus by genomic Southern blot-RFLP(restriction fragment length polymorphism) analysis did not reveal anyevidence of gene loss or gross rearrangement in the cell lines that didnot show prostasin expression, namely, MDA-MB435 (data not shown)Consideration of the possibility of prostasin gene-silencing byepigenetic mechanisms was also considered. Polysynthetic methylation ofcytosine residues in the 5′-CpG islands is often involved in long-termsilencing of certain genes during mammalian development and in theprogression of cancers. An examination of the prostasin promoter andexon I region sequence (GenBank accession number U33446) identified 28CpG dinucleotides in a segment defined by an Xho I site (vse number 374of U33446 or −1,048 relative to the transcription initiation site and aBamH I site (base number 1,649 of U33446 or +228 relative to thetranscription initiation site) (FIG. 9 a*) A GC enriched domaincontaining 2 consensus G/C boxes with the sequence GGGCGG was identifiedas encompassing the transcription initiation site. This GC enricheddomain extends from base number 1,101 to the BamH I site (1,649) as alength of 549 bp and has a GC content of 58.6%. The 549 bp GC-enricheddomain contains 19 CpG dinucleotides, but failed to qualify as a trueCpG island by the standards of Gardiner-Garden, M, and Frommer, M. CpGislands in vertebrate genomes. J Mol Biol 1987:196:261-82.

In all breast cancer cell lines and the NHMEC, XhoI/BamH I/Hha digestionyielded a 1,275 bp prostasin promoter band seen in the Xho I/BamH I/Idigestion (**FIG. 9 b upper panel) indicating that the −807 Hha Isite-CpG is homogenously methylated.

In the NHMEC digestion with Xho I/Bam H I/Aci I yielded a 1,072 bp, a727 bp and a 345 bp band, but no 1,275 bp band, indicating that the −320Aci I site-CpG is heterogeneously methylated but the +24 Aci I site CpGis homogenously unmethylated. In MCF-7, MDA-MB-453 and MDA-MB-231,digestion with Xho I/BamH I/Aci I yielded only the 1,072 bp band,indicating that the −320 Aci site CpG is homogenously methylated, butthe +24 Aci site CpG is homogenously unmethylated. In MDA-MB-435s,digestion with Xho I/BamH/Aci I yielded only the 1,275 bp band,indicating that both the −320 and the +24 Aci I site CpG's arehomogenously methylated.

In NHMEC and MCF-7 cells the 1,275 bp Xho I/BamH I band can be digestedby the methylation-insensitive Msp I as well as by the methylationsensitive Hpa II yielding an 951 bp band, indicating that the CpG at the−96 M/H restriction site is homogenously unmethylated. In MDA-MB-453,the 1,275 bp band can be fully digested by Msp I but only partially byHpa II indicating heterogenous methylation of the −96 M/H site CpG. InMDA-MB-231 and MDA-MB-435s, the 1,275 bp band was undigested by Hpa IIwhile having been completely digested by Msp I, indicating that the −96M/H site CpG is homogenously methylated.

Overall, in the prostasin-expressing cells the NHMEC is the leastmethylated in this region and the methylation is restricted to beyond−266 relative to the transcription initiation site. The prostasin genepromoter-exon I region in MCF-7 and MDA-MB-453 cells is methylated to ahigher extent than that in the NHMEC. In the MCF-7, methylation isrestricted to beyond −966, while in the MDA-MB-453, methylation isobserved at the −96 position and beyond. In the cells that do notexpress prostasin, the promoter-exon I region is the most heavilymethylated. While in MDA-MB-231 the methylation is still restricted tothe 5′flanking region, all CpG sites examined, including 1 of exon 1(+24 Aci I site CpG) were found to be homogenously methylated in theMDA-MB-435s cells. The results showed that the prostasin genepromoter-exon 1 region CpG methylation patterns correlate with theabsence of prostasin expression in the human breast cells examined.

Prostasin Expression Was Reactivated By Demethylation And HistoneDacetylase Inhibition In The Invasive And Metastatic Breast Cancer CellLines

Methylated promoter DNA may contribute to repression of gene expressionby interfering with the binding of transcription factors, or byinteracting with various methyl-CpG binding proteins. Such as the McCP 1complex, MeCP2 and the MBD's depending on cell type, sequence of themethylated regulatory region and binding of other transcription factors.Histone deacetylases are recruited by the methyl-CpG binding proteins toco-repress gene expression via chromatin restructuring. Histonedeacetylases act synergistically with DNA methylation in theco-repression, with DNA methylation being the dominant force for stablemaintenance of gene silencing in cancer. Treatment of cancer cells withthe demethylation agent 5-aza-2′-deoxycytidine (5-aza-2′dC) may restorea minimal expression for genes silenced by promoter methylation and thecombined treatment of 5-aza-2′-dC with the histone deacetylaseinhibitor, trichostatin A (TSA) may further stimulate the restoredexpression. Investigation of demethylation and/or histone deacetylaseinhibition could reactive prostasin gene expression in the MDA-MB-231and MDAA-MB-435s cells proceeded as follows:

After 8 days of treatment with the DNA methyltransferase inhibitor5-aza-2′-dC, a significant level of demethylation was observed in theprostasin promoter-exon 1 region in the MDA-MB-435s cells, as genomicDNA of this region in these cells was partially digested by Hha I at−807, Aci I at −320, BsaA I at −266 and Hpa H at −96 (***FIG. 9 c). The+24 Aci I was also partially digested in MDA-Mb-435s followingdemethylation (FIG. 9 c) Examination of total RNA of these cells byRT-PCR/Southern blot analysis following the 8-day 5-aza-2′-dC treatment,however, failed to detect prostasin mRNA expression (data not shown).

Investigation of the combined effect of treating the cells with5-aza-2′-dC and TSA, an inhibitor of histone deacetylase followed. InRT-PCR/Southern blot analysis, a time-dependant reactivated expressionof prostasin mRNA was observed in both the MDA-MB-231 and theMDA-MB-435s cells after a treatment with 5-aza-2′-dC for 24 hr followedby 95% ethanol (solvent for TSA) did not result in prostasin mRNAre-expression. A co-amplification of the β-actin message confirmed thequality and quantity of the total RNA used in each RT-PCR. When MCF-7and MDA-MB-453 cells were subjected to the same 5-aza-2′-dC/TSAtreatment and prostasin RT-PCR/Southern blot analysis procedures, noincrease of prostasin mRNA expression was observed for either cell line.

FIG. 9 a The solid horizontal line represents the promoter-exon I regionof the human promstasin gene (Genbank U33446) The Xho I site is locatedat Base 1,649 of the U33446 sequence respectively. The methylationsensitive restriction sites used for differential methylation analysis,Hha (615/−807), Aci I (1,102/−320 and 1,445/+24), BsaA I (1,156/−266)and Msp l/Hpa II (1,326/−96) are indicated by arrows. Four additionalAci I sites and a second dMsp Ihpa Ii site are present between the +24Aci I site and the Bam H I sies but not shown in the figure since theydo not affect the results of the genomic DNA Southern blot analysis. Thetranscription initiation site is indicated by the triangle on anextended vertical bar. The vertical bars map the location of the 28 CpGdinucleotides identified in this region. A 549 bp GC-enriched domain isindicated by the filled rectangular box. The extended vertical bas withopen square boxes indicate the locations of the consensus G/Cboxes. Thelocation of the probe used in the genomic DNA Southern blot analysis isshown by the open rectangular box.

FIG. 9 b. Panels of genomic DNA Southern lot analysis results are asindicated for each cell type. Restriction endonucleases used in eachdigestion mixture are indentified as follows: X, Xho I, B, Bam H I, Hh,Hha I, A, Aci I, Bs, BsaA I, M, Msp I, H, Hpa II. Hha I, Aci I, BsaA Iand Hpa II only cut unmethylated DNA while Msp I, an isoschizomer of HpaIi, cuts unmethylated or methylated DNA. Genomic DNA (10 μg) from eachcell type was cut with X/B or with XIB/M to serve as controls for thedetection of differential methylation. In the upper panel, prostasinpromoter DNA will yield a 1,275 bp X/B fragment. When Hha I was added tothe X/B digestion mixture a fragment of 1,037 bp was expected if the−807 Hha I site CpG was not methylated. When Aci I was added to the X/Bdigestion mixture, 3 smaller bands might be expected depending on CpGmethylation states at −320 and +24 Aci I sites. They are 1,072 bp(fromXho I to +24 Aci I) 727 bp (from Xho I to −320 Aci I) and 345 bp (from−320 Aci to +24 Aci I) in length, respectively. When BsaA I was added tothe X/B digestion mixture, 2 smaller bands might be expected dependingon CpG methylation state at the −266 Bsa A I. They are 782 bp (from XhoI to BsaA I) and 493 bp (from BsaA I to BamH I) in length, respectively(indicated by asterisks in the figure). In the lower panel, the upperarrow points to the X/B fragment, while the lower arrow points to thefragment that is generated by Msp I, regardless of DNAmethylation, or byHpa LI, only when DNA is unmethylated.

FIG. 9 c Cells treated with 5-aza-2′dC for 8 days were harvested forgenomic DNA isolation and southern blot analysis as described. Thearrowhead indicates the 1,037 bp Hha I-BamH I fragement when the −807 haI site was cut by enzyme following DNA demethylation.

Example 2

The same methylated sites investigated in example I above in breastcancer cell lines, were investigated in human prostate cancer cell linesand human prostate epithelial cells. All methods were the same, exceptas follows:

Cell Culture Maintenance

A normal human prostate epithelial cell (PrEC) primary culture (PrECprimary culture (Catalog Number CC-2555) was obtained from Clonetics(San Diego, Calif.) and maintained in the supplied medium (prostrateepithelial cell basal medium, containing bovine pituitary extract,hydrocortisone, human epidermal growth factor, epinephrine,transferring, insulin, retinoic acid, tri-iodothyronine, gentamicin, andamphotericin). The culture was kept at 37° C. with 5% CO₂ and used forexperiments at the overall passage.

Human prostate cancer cell lines LNCAP, DU-145 and PC-3 were obtainedfrom the American Type Culture Collection (ATCC, Manassas, Va.). ThePC-3 cells were maintained in F-12K medium supplemented with 10% fetalbovine serum (FBS) while the LNCaP and the DU-145 cells were maintainedin RPMI-1640 medium supplemented with 10% FBS and 1 mM sodim pyruvate,and all were kept at 37° C. with 5% CO₂.

Demethylation Of Prostasin Gene Promoter And Reactivation Of ProstasinExpression

Du-145 and PC-3 cells were seeded in 60-mm dishes at 80% confluence andcultured in the presence of 500 nM 5-aza-2′-dC for 24 hours and werethen treated for an additional 24 hours with either 1 μM trichostatin A(TSA, Sigma-Aldrich Co.) or an equal volume of 95% ethanol used todissolve TSA. TNA was isolated for prostasin-specific RT-PCR/Southernblot analysis as described in example 1.

As in example 1, the prostate cells were subjected to Southern blotanalysis. In all three prostate cancer cell lines and the PrEC,XhoI/BamH I/Hha I digestion yielded a 1,275 bp prostasin promoter bandseen in the XhoI/BamH I/Hha I digestion (FIG. 10, upper panel),indicating that the −807 Hha site-CpG is homogeneously methylated.

In the PrEC, digestion with Xho I/Bam H I/Aci I yielded a 1,072 bp, a727 bp and a 345 bp band, but no 1,275 bp band indicating that the −320Aci I site CpG is heterogeneously methylated but the +24 Aci site CpG ishomogeneously unmethylated. In LNCaP and Du-145, digestion with XhoI/BamH I/Aci I ielded only the 1,072 bp band indicating that the −320Aci I site-CpG is homogenously methylated but the +24 Aci I site CpG ishomogeneously unmethylated. In PC-3, digestion with Xho I/BamH I/Aci Iyielded the 1,072 bp band and to a lesser extent the 1,275 band,indicating that the −320 Aci I site-CpG is homogensously methylated,while the +24 Aci I site pG is heterogeneously methylated.

In the PrEC and LNCaP digestion with Xho I/BamH I/BsaA I yielded a 782bp and a 493 bp band, indicating that the −266 BsaA I site CpG ishomogeneously unmethylated. In DU-145, digestion with Xho l/BamH I/BasAI yielded the 782 bp and the 493 bp bands, but also the 1,275 bp band,indicating that the −266 BsaA I site-CpG is heterogeneously methylated.In PC-3, digestion with Xho/BamH I/BsaA I yielded only the 1,275 bpband, indicating that the −266 BsaA I site-CpG is homogeneouslymethylated.

In the PrEC and LNCaP cells, the 1,275 bp Xho I-BamH I band can bedigested by the methylation-insensitive Msp I as well as by themethylation-sensitive Hpa II, yielding a 951 bp band (FIG. 10, lowerpanel) indicating that the CpG at the −96 M/H restriction site ishomogeneously unmethylated. In DU-145 and PC-3, the 1,275 bp band can befully digested by Msp I but only partially by Hpa II (FIG. 10, lowerpanel) indicating heterogeneous methylation of the −96 M/H site Cpg.

The PrEC express both the prostasin mRNA and protein and is the leastmethylated in the prostasin promoter-exon 1 region (methylation is onlyobserved at beyond −266 relative to the transcription initiation site.The prostasin gene promoter-exon 1 region in LNCaP cells is methylatedto a higher extent than that in the PrEC. In LNCaP, which also expressboth the prostasin mRNA and protein, methyltion is only observed atbeyond −96. In DU-145, which does not express either the prostasinprotein or mRNA, the promoter-exon 1 region is the most heavilymethylated. While in DU-145, the methylation is limited to the 5flanking region, all CpG sites examined, including one of exon 1 (+24Aci I site-CpG) show heterogeneous to homogeneous methylation in thePC-3 cells. The prostasin gene promoter-exon region CpG methylationpatterns correlate with the absence of prostasin expression in the humanprostate cells.

Table I details the methylation state of the prostate cells. As shownwith the breast cancer cells the methylation state of the prostasinpromoter region may be examined for a potential diagnostic applicationto indicate prostate cancer invasiveness. For example, the prostasingene promoter methylation state at the −96M/H CpG site, for example, isseen methylated in invasive cell types.

TABLE 1 CpG methylation in the prostasin gene promoter-exon1 region inhuman prostate cells. Pro- stasin Msp1/ Cell Expres- Hha 1 Aci 1 BsaA 1Hpa 1 1 Ac1 1 Type sion (−807) (−320) (−266) (−96) (+24) PrEC Yes MethylHetero- Un-Methyl Un- Un- methyl Methyl Methyl LNCaP Yes Methyl MethylUn-Methyl Un- Un- Methyl Methyl DU-145 No Methyl Methyl Hetero- Hetero-Un- Methyl Methyl Methyl PC-3 No Methyl Methyl Methyl Hetero- Hetero-Methyl Methyl Table Legend: Methyl = homogeneously methylatedHetero-Methyl = heterogeneously methylated Un-Methyl = homogeneouslyunmethylated

In investigating whether methylation in the promoter is causal to thelack of prostasin gene expression in the DU-145 and PC-3 cells, as inthe breast cancer cells, treatment of these cell lines for 8 days withthe DNA methyltransferase inhibitor 5-aza-2′-dC resulted in significantlevel of demethylation but no prostasin mRNA expression was detected inan RT-PCR/Southern blot analysis of the DNA demethylated cells.Treatment of the cells with the combination of 5-aza-2′-dC andtrichostatin A (TSA) an inhibitor or histone deacetylase howeverrestored prostasin mRNA expression in DU-145 and PC-3 cells (data notshown) When investigating whether the prostasin protein was expressed inthe 5-aza-2′dC/TSA treated cells by western blot analyis, the result wasnegative. It appears that although promoter DNA methylation is causal toabsence of prostasin expression in the invasive prostate cancer cells,the reactivated expression represents only the basal expression, similarto what was observed in the breast cancer cells.

Examples I and II demonstrate that the methylation patterns of the samespecific sites in both breast cancer and prostate cancer provide markersfor invasiveness.

While the invention has been described, disclosed, illustrated and shownin various terms of certain embodiments or modifications which it haspresumed in practice, the scope of the invention is not intended to be,nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

1. A method for determining whether human prostate or breast carcinomasare non invasive, comprising sampling human breast or prostatecarcinomas and determining whether the prostasin gene promoter DNA ismethylated at bp 1,156/−266, wherein the absence of methylationindicates a non-invasive carcinoma.
 2. The method, as in claim 1 whereinthe sampling of the carcinoma tissue includes the step of separating thehuman carcinoma tissue from neighboring normal tissue using a lasercapture micro-dissection.
 3. The method, as in claim 1, wherein the stepof determining gene promoter DNA methylation patterns includes applyingprostasin-promoter-specific oligonucleotide primers in a PCR todetermine the prostasin gene promoter DNA methylation patterns in thesampled tissue.
 4. A method for determining which cancer patients do notrequire chemotherapeutic treatment, comprising taking a biopsy of aprostate or breast tumor, and determining the non-invasiveness of thecarcinoma tissue based on the absence of methylation of the prostasingene promoter DNA at bp 1,156/−266.
 5. A method for determining whetherhuman prostate carcinomas are non invasive, comprising sampling humanprostate carcinomas and determining whether the prostasin gene promoterDNA is methylated at bp 1,156/−266, wherein the absence of methylationindicates a non-invastive caarcinoma and the presence of methylationindicates invasive carcinoma.
 6. The method of claim 5 wherein thesampling of the carcinoma tissue includes the step of separating thehuman carcinoma tissue from neighboring normal tissue using a lasercapture micro-dissection.
 7. The method of claim 5, wherein the step ofdetermining gene promoter DNA methylation patterns includes applyingprostasin-promoter-specific oligonucleotide primers in a PCR todetermine the prostasin gene promoter DNA methylation patterns in thesampled tissue.